Novel insights into the role of bisphenol A (BPA) in genomic instability

Abstract Bisphenol A (BPA) is a phenolic chemical that has been used for over 50 years in the manufacturing of polycarbonate and polyvinyl chloride plastics, and it is one of the highest volume chemicals produced worldwide. Because BPA can bind to and activate estrogen receptors, studies have mainly focused on the effect of BPA in disrupting the human endocrine and reproductive systems. However, BPA also plays a role in promoting genomic instability and has been associated with initiating carcinogenesis. For example, it has been recently shown that exposure to BPA promotes the formation of single stranded DNA gaps, which may be associated with increased genomic instability. In this review, we outline the mechanisms by which BPA works to promote genomic instability including chromosomal instability, DNA adduct formation, ROS production, and estrogen receptor (ER) activation. Moreover, we define the ways in which BPA promotes both carcinogenesis and resistance to chemotherapy, and we provide critical insights into future directions and outstanding questions in the field.


Introduction
A phenolic chemical which has been used for over 50 years in the manufacturing of polycarbonate plastics, epoxy resins, thermal paper and polyvinyl chloride plastics, bisphenol-A (BPA) ranks in the top 2% of high-production-volume chemicals, with 5-6 billion pounds produced annually worldwide ( 1 ).BPA is an estrogenic compound which has been shown to bind to the nuclear estrogen receptor (ER) alpha and beta as well as the membrane-associated GPR30 ( 2 ,3 ).Because of its mass production, exposure to BPA is extremely prevalent.For example, a study in 2004 revealed that from a sample of 2517 individuals over the age of 6, 93% were found to have detectable levels of BPA in their urine ( 1 ).As of 2021, the median creatinine-adjusted urinary BPA concentration in adult humans was estimated to be 1.76 μg / g (95% PI: 0.79-2.73),and the pooled estimate for serum BPA was 1.75 μg / l (95% PI: 0-10.58) ( 4 ).
The main mechanism of exposure to BPA is through the diet, as BPA is capable of leaching through plastic products into proximal foods and liquids ( 5 ).BPA can also enter the body via inhalation of dust or fumes from burned plastics.Interestingly, populations with particularly high levels of BPA exposure include infants, especially those in neonatal intensive care units, and dialysis patients, due to leaching of BPA from medical equipment into fluids entering the body (6)(7)(8).Additionally, a 2014 study revealed that U.S. manufacturing workers in factories producing BPA containing products exhibited urinary BPA levels about 70 times greater than that of the general population, mainly via inhalation and dermal absorption ( 9 ).
Questions regarding the safety of BPA began to emerge around the year 2003, when a paper describing potential harm from BPA exposure was published ( 10 ).After accidental exposure to BPA from animal cages, the oocytes of female mice were shown to exhibit a sudden, spontaneous increase in meiotic disturbances, including aneuploidy ( 10 ).Since then, exposure to BPA has been associated with a wide variety of adverse health effects such as abnormalities in reproductive organ function, placental dysfunction, precocious puberty, and neurological impairment ( 11 ,12 ).For example, BPA exposure has been shown to upregulate the expression of gonadotropin releasing hormone (GnRH) and follicle stimulating hormone (FSH), hormones involved in the regulation of the menstrual cycle and spermatogenesis (13).In addition, BPA has also been shown to inhibit the synthesis of testosterone and increase the expression of aromatase and estrogen in mice pups ( 13 ).
Despite this evidence, the US Environmental Protection Agency (EPA) in conjunction with the Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC) and the National Institute of Environmental Health Sciences (NIEHS) have not taken regulatory action in regards to BPA, stating that these studies are insufficient for risk assessment due to either the dose used, flaws in some of the study designs, scientific uncertainty concerning the relevance to health and ecological hazard of the reported effects, and the inability of other researchers to reproduce the effects in standardized studies ( 14 ).In 2012, these organizations collaborated to form the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) program with the goal of studying the full range of potential health effects from exposure to BPA.While BPA exposure increased the incidence of pathological lesions such as mammary gland adenocarcinoma and follicular ovarian cysts, the study concluded that these effects were not dose responsive, sometimes occurring in only one low or intermediate dose group and did not demonstrate a clear pattern of consistent responses within or across organs ( 15 ).
As of today, the EPA and FDA have established a safe reference dose (RfD) of BPA for humans at 50 μg / kg / day.This value is based on a 1982 study by the National Toxicology Program (NTP) that concluded that while pharmacological doses of BPA induce some cancers in both male and female adult rodents, it is not a robust carcinogen at doses relevant to human exposure ( 16 ).Important to consider however, is that BPA follows a non-monotonic dose-response curve, meaning that both low and high doses can produce similar phenotypes ( 17 ,18 ).While concentrations utilized in animal model experiments tend to hover around the RfD, cell culture experiments typically use a higher concentration of BPA, albeit for a much lesser period of time (Table 1 ).Whether higher levels of exposure for a shorter duration mimic the lifetime exposure in most humans is still unknown.Given that BPA, like most estrogenic chemicals, does not follow a typical linear dose response relationship, it is imperative to consider the relevance of these studies and how they may translate to human health.
Moreover, while the main focus of BPA on human health has revolved around metabolic and sexual dysfunction, recent literature has uncovered a role for BPA as a DNA damaging agent, mutagen, and potential carcinogen.For example, in animal models, in utero exposure to BPA has been shown to consistently alter mammary gland architecture (19)(20)(21).Moreover, perinatal exposure to BPA has been associated with an age dependent increase in mammary epithelial cell proliferation ( 22 ).While animal studies indicate a role for BPA as a potential carcinogen, reviewed in ( 23 ,24 ), many still yield conflicting results and the physiologic relevance of BPA concentrations remains a question.This review aims to outline both mechanisms of BPA in promoting genomic instability as well as cellular proliferation and carcinogenesis and propose open questions and research directions in this critical field.An overview of the studies discussed, listing the concentration of BPA and the model system used, is presented in Table 1 .

Mechanisms of BPA-induced genomic instability
A characteristic of most cancers, genomic instability refers to the increased frequency of acquired mutations, structural rearrangements, and copy number alterations in the genome.Caused by dysfunctional maintenance of the genome or exposure to carcinogens, genomic instability is primarily categorized by defects in DNA repair, DNA replication, cell cycle control or chromosome segregation.When genomic integrity is impaired, the potential for carcinogenesis greatly increases, highlighting the importance of maintaining genomic stability ( 25 ).
While not currently classified as a carcinogen, BPA has been shown to damage DNA, leading to genomic instability and potentially carcinogenesis ( 26 ).Potential mechanisms through which this occurs, discussed in this review, include impairment of chromosomal segregation, DNA adduct formation, single and double stranded DNA break formation, reactive oxygen species formation, and estrogen receptor-mediated DNA damage.

Impaired chromosome segregation
As previously stated, one of the mechanisms of promoting genomic instability is deregulation of chromosomal segregation.This is primarily maintained via the mitotic spindle assembly checkpoint (SAC), a surveillance mechanism that promotes proper segregation of chromosomes during anaphase.Impairment of this process leads to gains, losses, or rearrangement of genomic material with the potential to cause defects in birth and development and promote carcinogenesis ( 27 ).
Highlighting one of the ways in which BPA can promote genomic instability, a 2003 study revealed that exposure to BPA adversely affects chromosome segregation, promoting mitotic aneuploidy in female mice ( 10 ).After accidental exposure to BPA from damaged cages, it was found that exposure to low doses of BPA ranging from 20 to 100 ng / g disrupted meiosis of female mice in a dose dependent manner ( 10 ).Additionally, a study from 2010 further characterized the role of BPA in impairing chromosomal segregation, revealing that BPA exposure impedes chromosome synapsis and disrupts progression of meiotic double strand break repair (DSBR) in a C. elegans model ( 28 ).This study showed that exposure to BPA impairs oogenesis, resulting in elevated levels of sterility and embryonic lethality.Moreover, the ability of BPA to act as a xenoestrogen resulted in a germline specific down-regulation of DSBR genes such as MRE11, hindering the maintenance of genomic integrity during meiosis ( 28 ).While the effects of BPA on mitotic progression in the context of ER activation remain primarily unknown, it has also been shown that BPAmediated ER activation promotes enhanced activation of the Src / Raf / Erk pathway, leading to chromosome malsegregation ( 29 ).Because ER activation promotes cellular proliferation, it is important to uncover whether BPA-mediated ER activation hinders mitotic progression or results in suppression of checkpoint control pathways, leading to further chromosomal instability.
In an attempt to further define the mechanisms in which BPA exposure leads to genomic instability and carcinogenesis, a study by Kim et al., investigated the effects of BPA on mitosis progression, revealing that BPA interferes with the attach-ment of spindle microtubules to kinetochores ( 30 ).By disturbing spindle attachment and activating the SAC, BPA prolongs mitotic progression.Additionally, BPA perturbs the localization of microtubule associated proteins, HURP and TPX2.BPA also generates a multipolar spindle by inducing over duplication of the centriole and premature disengagement.This is shown to occur in an estrogen receptor (ER) independent manner ( 30 ).

DNA damage and repair
When a DNA base is chemically modified, the damaged base must be repaired or excised in order to ensure faithful replication of the DNA and prevent mutagenesis.This can occur through processes such as direct reversal, base excision repair, or nucleotide excision repair in which the modified base is removed and subsequently replaced by a DNA polymerase.However, some adducts may escape DNA repair, and persist into S-phase ( 31 ).
When a cell's replication machinery encounters a DNA adduct, this can lead to replication stress, a major cause of genomic instability.These adducts can either be replicated through, via a mechanism known as translesion DNA synthesis (TLS), or the DNA synthesis downstream of the adduct can be reprimed by PrimPol, a polymerase with priming capability ( 32 ).Replicating over the base may lead to nucleotide misincorporation, in which a mutation of the base opposite the lesion or at the position corresponding to the lesion becomes fixed during the first and second round of replication, respectively.On the other hand, repriming will induce the formation of a single stranded DNA (ssDNA) gap, which when transmitted to the next cycle of DNA replication, leads to collapse of the replication fork, resulting in further genomic instability ( 32 ).
BPA has been shown to form adducts with DNA, specifically after oxidation into bisphenolo -quinone (BPAQ) ( 33 ).Once BPA enters the body, it is metabolized and inactivated in the liver by the addition of glucuronide, forming Bisphenol A glucuronide.This conjugate has a half-life of 2 h and is primarily secreted into the urine ( 33 ).However, BPA can also be deconjugated and reactivated by β-glucuronidase, an enzyme found in tissues such as the placenta and the liver ( 34 ,35 ).This reactivated form of BPA is then able to form DNA adducts (Figure 1 ) (36 ).
BPA has been shown to form DNA adducts in human breast cancer cells as well as in vivo in murine liver and mammary tissue ( 37 ,38 ).A study in 2021 by Hu et al. investigated the effects of BPA adduct formation on the human genome, identifying patterns of genome-wide point mutations and genomic rearrangements associated with BPA exposure ( 39 ).Preferred sites of mutagenesis included regions at or near guanines, confirming the tendency of BPA to form guanine adducts.Moreover, BPA treatment resulted in a high frequency of insertions and deletions (InDels), DNA DSBs and structural variants, suggesting that BPA adducts result in DNA breaks during replication or are replicated through by translesion synthesis polymerases.Finally, it was shown that genomic signatures associated with BPA-mediated DNA damage exhibit high similarity to those of digestive and urinary tumors, both of which are associated with increased environmental exposure to BPA ( 39 ).While this study suggested that BPA adducts are replicated through by TLS polymerases, whether this occurs remains to be explored.Additionally, it is unknown which TLS polymerases engage at BPA-DNA lesions.
BPA has also been shown to promote the formation of single stranded and double stranded DNA breaks in replicating cells, presumably through its ability to form DNA adducts.In human hepatoma cell lines, BPA was shown to induce the formation of DNA breaks after 24 h, indicating that DNA replication must occur before breaks can be detected ( 40 ).Moreover, BPA has also been shown to promote a dose dependent increase in single and double stranded DNA breaks in human peripheral blood mononuclear cells (PBMCs) which were unable to be totally repaired ( 41 ).
Recently, our laboratory showed that BPA exposure causes the formation of nascent strand single stranded DNA (ssDNA) gaps which, in addition to homology directed repair (HDR), are fundamental to the sensitivity of BRCA deficient cells to genotoxic agents such as cisplatin and PARP inhibitors (42)(43)(44)(45).When ssDNA accumulates, deposits of RPA, a protein that binds to and protects ssDNA, become depleted due to increased demand, and cells become increasingly reliant on post replicative mechanisms of DNA repair.The accumulation of ssDNA gaps leads to increased formation of dsDNA breaks, which are unable to be repaired in cells lacking efficient homologous recombination (HR) ( 46 ).Ultimately, the increase in DSB formation in these cells leads to genomic instability and cell death, potentially underlying the sensitivity of BRCA deficient cells to ssDNA gap inducing agents ( 47 ).
Our recent study revealed that BPA promotes the accumulation of nascent strand ssDNA gaps ( 45 ).Moreover, we showed that these gaps are expanded bidirectionally by the MRE11 and EXO1 nucleases, and both expansion of the gap by the exonucleolytic activity of MRE11 and EXO1 as well as cleavage of the template strand by the endonucleolytic activity of MRE11 is required for conversion of the BPA induced ssDNA gap to a dsDNA break.Left unrepaired, the accumulation of DSBs will likely promote an increase in chromosomal structural variations, further increasing genomic instability (Figure 2 ).We also showed that exposure to BPA decreases cellular viability, which can be rescued by knockdown of EXO1, suggesting that exonucleolytic gap processing leads to cytotoxicity ( 45 ).
How BPA-mediated ssDNA gaps are repaired to avoid their conversion to DSBs is still unclear.As mentioned in the previous section, filling of these gaps or replication over BPA adducts may occur through TLS.
While not yet shown, we hypothesize that formation of BPA adducts can also promote PCNA ubiquitination at stalled replication forks, an event which is required for TLS.This recruits TLS polymerases which can bypass the BPA adduct (Figure 2 ).Our model also implies that after the initial adduct bypass, REV1 and Pol ζ polymerases extend the DNA, resulting in gap filling and allowing ligation.Because TLS polymerases are error prone, this process may result in point mutations.As shown by Hu et al., exposure to BPA results in both structural variants and point mutations, supporting the idea that these adducts can be replicated through by TLS or form ssDNA gaps which, when left unrepaired, result in endonucleolytic DSB formation ( 39 ,45 ).

Reactive oxygen species formation
Formed as a byproduct of metabolism, reactive oxygen species (ROS) serve as a significant source of endogenous DNA damage, resulting in DNA base modifications, single-and doublestrand breaks, and apurinic / apyrimidinic lesions ( 48 ).In turn, upon DNA damage, further induction of ROS is promoted via histone H2AX accumulation, shown to be mediated through Nox1 and the Rac1 GTPase ( 49 ,50 ).
Exposure to BPA has also been shown to promote the accumulation of ROS and enhance signaling of oxidative stress mediated signaling pathways ( 51 ).For example, BPA has been shown to decrease cellular viability via the depletion of the intracellular level of ATP and alterations in human periph- eral blood mononuclear cell (PBMC) size and granulation ( 51 ).Even moderate reductions in levels of intracellular ATP are enough to promote oxidative stress ( 52 ).Moreover, BPAmediated increases in ROS levels exhibit both damage to proteins and lipids in PBMCs ( 53 ).This increase in ROS formation promotes an increase in both single and double stranded DNA breaks as well as an increase in the expression of the DNA damage-associated proteins p53 and p-Chk2 ( 54 ).BPA has also been shown to induce oxidative stress by decreasing the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GSH-Px) and increasing hydrogen peroxide and lipid peroxidation in the liver and epididymal sperm of rats ( 55 ).
While both BPA-driven adduct formation and ROS production can cause single and double stranded DNA breaks, the mutational signatures of the two have yet to be differentiated.Further avenues for investigation include distinguishing the mutational spectra of BPA-mediated DNA damage based on the mechanism in which it occurs.While it is possible that these signatures overlap, identifying mechanisms of BPA-mediated DNA damage may help to uncover if different routes of exposure cause damage in different ways.If so, this would greatly assist in guiding the development of mechanisms to minimize harm from BPA exposure.Additionally, whether repair of BPA adduct-derived ssDNA gaps occurs via the same mechanism as repair of damage from ROS is still unclear.Understanding which origins of damage promote which mechanism of DNA damage tolerance and repair is important to fully outline the effect of BPA on genomic instability.

Estrogen receptor mediated DNA damage
Also important to consider is the role of BPA as a xenoestrogen and how its activity as an endocrine disruptor affects DNA damage and repair.Activation of the estrogen receptor (ER) has been shown to promote genomic instabil-ity.For example, estrogen (E2) mediated ER activation has been shown to enhance the formation of co-transcriptional R-loops, which have been linked to DNA damage ( 56 ).Activation of the ER has also been shown to increase the transcription of genes involved in the repair of double strand DNA breaks such as MRE11, RAD50 and PALB2, and ER-mediated transcription has also been shown to induce the formation of cell-cycle dependent dsDNA breaks ( 57 ,58 ).Intriguingly, since MRE11 also expands BPA-induced ssDNA gaps, increased MRE11 levels after ER activation may further promote gapinduced genomic instability.ER activation also promotes cellular proliferation through upregulation of protooncogenes and growth factors such as c-myc, and reliance on ER signaling is a critical regulator of cellular proliferation and survival in a majority of breast cancers ( 59 ).
In addition to the role of E2 in inducing DNA damage via ER activation, BPA has also been shown to promote genomic instability through its estrogenic activity.For example, BPA exposure promotes the formation of dsDNA breaks to a greater extent in ER-expressing cells than ER-negative cells ( 60 ).As previously mentioned, BPA-mediated ER activation promotes enhanced activation of the Src / Raf / Erk pathway, leading to chromosome mis-segregation and increased micronuclei formation ( 29 ).

A dditional mec hanisms of BPA in cancer etiology and treatment
Despite scientific evidence suggesting that BPA promotes DNA damage and carcinogenesis, BPA is not classified as a carcinogen by the Environmental Protection Agency, primarily due to lack of consistency between studies ( 15 ,61 ).Moreover, as chemoresistance remains a key problem in the clinic, understanding of carcinogenic risk factors and how they relate to tumor formation remains essential to enhancing treatment de-Figure 2. Proposed mechanisms of BPA-induced genomic inst abilit y during carcinogenesis.When the replication fork encounters a BPA adduct, repriming is initiated downstream of the lesion by PrimPol, leaving behind a ssDNA gap.This gap is then expanded bidirectionally by MRE11 and EXO1.If left unrepaired, the template strand opposite of the gap is nicked by the endonuclease activity of MRE11, forming a dsDNA break which can promote the accumulation of chromosomal aberrations.We h ypothesiz e that BPA adducts can also be replicated through via TLS.TLS polymerases are capable of filling the gap, but they are also error prone, promoting the formation of point mutations.
cisions.The following section will focus on mechanisms in which BPA promotes aberrant cellular proliferation as well as evidence linking BPA exposure to chemotherapy resistance.

Transcriptional effects
One way in which BPA may exert a carcinogenic effect is through transcriptional regulation.For example, BPA exposure has been shown to upregulate the expression of c-MYC in estrogen receptorα (ER α)-negative mammary cells, promoting DNA damage and cellular proliferation ( 62 ).This is thought to occur via induction of HDAC6 expression which, in turn, downregulates the tumor suppressor gene PTEN, resulting in upregulation of c-MYC expression ( 63 ).Silencing of c-myc reduces BPA-mediated DNA damage and cellular proliferation, suggesting that expression of c-MYC is essential for regulating effects of BPA on DNA damage and proliferation in mammary cells ( 62 ).BPA has also been shown to induce the expression of the GPER (G-protein coupled estrogen receptor) target genes c-FOS, EGR-1 and CTGF through the GPER / EGFR / ERK transduction pathway in SKBR3 breast cancer cells and cancer associated fibroblasts (CAFs) ( 64 ).Similar to c-MYC, the proliferative effects of BPA were reduced when GPER expression was silenced, indicating that BPA-mediated cellular proliferation relies on upregulation of both GPER and c-MYC ( 64 ) (Figure 3 A).
Moreover, BPA has also been shown to induce activation of the mammalian target of rapamycin (mTOR) pathway ( 65 ).In both human high-risk donor breast epithelial cells (HRBEC) and T47D breast cancer cells, BPA exposure promotes increased expression of proliferation-initiating gene products such as proliferating cell nuclear antigen (PCNA), cyclins, CDKs and phosphorylated Rb ( 65 ) (Figure 3 A).BPA exposure also promotes reduced expression of proapoptotic regulators of the cell cycle including, p53, p21 WAF1 and BAX (Figure 3 B).BPA was also shown to extend the proliferation potential of 6 independent HRBEC cultures ( 65 ).Continuous growth of one of the HRBEC cultures was even maintained after BPA removal, suggesting that BPA promotes genomic instabilitydriven hyperproliferation ( 65 ).
Additionally, BPA plays a role in the dysregulation of genes associated with physiological development and cancer pathogenesis.For example, BPA has been shown to induce the expression of HOXB9, a homeobox-containing gene that plays a key role in mammary gland development ( 66 ).While HOXB9 is transcriptionally regulated by estradiol (E2), the estrogenresponse-elements (EREs) in the HOXB9 promoter can also be stimulated in the presence of BPA, modifying chromatin methylation, and promoting gene activation ( 66 ) (Figure 3 C).
In addition to mammary development, BPA has also been shown to activate catenin-beta-1 (CTNNB1), a key gene indicated in lymphoma pathogenesis ( 67 ).Activation of CTNNB1 by BPA results in an increase in DNA SSB and DSB damage as well as cell cycle arrest in the G2 / M phase for DNA repair ( 67 ).

Cellular proliferation and tumorigenesis
In addition to promoting transcription of genes implicated in cancer pathogenesis, BPA exposure has also been directly associated with aberrant cellular proliferation and transformation.For example, BPA alone, or in combination with di(2-ethylhexyl) phthalate (DEHP) has been shown to induce mammary gland hyperplasia and promote proliferation of ductal epithelial cells, resulting in an increase in lobule and acini formation ( 68 ).BPA exposure also reduces tumor latency, enhancing the susceptibility of mammary tissue to carcinogenesis.These findings were associated with upregulation of ESR1 and HDAC6 which led to a further activation of c-MYC, also shown to be essential to BPA-induced mammary cell proliferation ( 62 ,68 ) (Figure 3 A).
Moreover, fetal mice exposed to BPA have been shown to exhibit increased susceptibility to DMBA-induced tumor formation.BPA was also shown to promote established tumor growth in mice injected with MCF-7 human breast cancer cells which was reversed by tamoxifen treatment ( 69 ).These findings indicate BPA's role in promoting cancer progression through both molecular alteration of fetal glands and estrogen receptor-dependent cell growth.Low doses of BPA corresponding to 25 μg BPA / l were also shown to promote mammary tumorigenesis and metastasis in MMTV-erbB2 mice ( 70 ).Decreased tumor latency and increased tumor burden in the presence of BPA was associated with increased phosphorylation of erbB2, erbB3, insulin-like growth factor 1 receptor, and Akt in the mammary gland ( 70 ).
BPA has also been shown to promote the self-renewal of stem-progenitor cells, specifically those in the human prostate.BPA exposure was found to induce phosphorylation of Akt and Erb as well as increase the expression of stem-related genes such as TBX3 and NANOG ( 71 ) (Figure 3 D).Moreover, continuous exposure to BPA increased highgrade prostate intraepithelial neoplasia and adenocarcinoma in human prostate epithelial grafts composed of prostate stemprogenitor cells ( 71 ).
In addition, BPA has been shown to promote both the proliferation and migration of T-29 human colon adenocarcinoma cells via phosphorylation of the extracellular signalregulated kinase (ERK) ( 72 ).BPA also reduced E-Cadherin expression, promoting epithelial to mesenchymal transition, and increased 5-HT3 receptor expression, a key mitogenic factor ( 72 ) (Figure 3 E).Moreover, BPA exposure has been shown to promote excessive ROS production, subsequently activating the HIF-1 α/ VEGF / PI3K / AKT axis and promoting the growth and migration of human colon cancer cells ( 73 ).BPA has also been shown to promote a dose-dependent increase in hepatic tumor incidence in adult mice following perinatal exposure ( 74 ).
While this evidence suggests that BPA may function as a direct carcinogen, other studies have indicated that BPA may promote carcinogenesis via epigenetic modifications.For example, BPA has been shown to alter DNA methylation patterns in genes responsible for prostate development.After BPA exposure, areas of hypomethylation were identified in a 5flanking CpG island of the phosphodiesterase type 4 variant 4 (PDE4D4) gene ( 75 ).This region is typically gradually hypermethylated with age in the human prostate.However, hypomethylation and increased PDE4D4 expression is often observed in prostate cancer formation, indicating a role for BPA in prostate cancer pathogenesis ( 75 ).Additionally, a study by the Dolinoy group investigated the effects of perinatal BPA exposure on longitudinal 5-hmC patterns at imprinted regions of the genome, showing that differentially hydroxymethylated regions of the murine genome exhibit consistent patterns of differential 5-hmC by developmental BPA exposure that persisted throughout adulthood ( 76 ).Further understanding of the interplay between BPA's role as a genotoxic agent and an epigenetic modifier is essential to fully outline its role in promoting carcinogenesis.

Chemotherapy resistance
While great advancements have been made in the way cancer is treated, resistance to chemotherapy remains a key clinical problem ( 77 ).In cancer patients receiving traditional chemotherapeutics or novel targeted drugs, multidrug resistance is responsible for over 90% of patient deaths ( 78 ).While chemotherapeutic resistance is primarily thought to be due to genetic changes involving the agent itself, exposure to environmental agents such as BPA can also promote resistance to treatment.For example, co-exposure of BPA with camptothecin (CPT), a topoisomerase inhibitor, was found to decrease the burden of Top1-DNA adducts as well as chromosomal aberrations and DNA strand break formation in mouse embryonic fibroblasts (MEFs) ( 79 ).Increased cellular survival due to BPA exposure was associated with wide-spread compaction of chromatin and loss of nuclear volume, reducing the accessibility of DNA to Top1, and inhibiting the function of CPT ( 79 ).
Moreover, BPA has also been shown to interfere with the function of the anthracycline doxorubicin, a topoisomerase II inhibitor.BPA exposure upregulated the expression of bclxl, a critical anti-apoptotic protein and reduces the sensitivity of Hep-2 and MRC5 cells to doxorubicin ( 80 ).These studies suggest that, while BPA typically promotes genomic stability and aberrant cellular proliferation via mechanisms described above, it may also directly impact the mechanism of action of topoisomerase inhibitors.This indicates the importance of outlining the function of BPA as it relates to genomic instability so that informed treatment decisions can be made, specifically as related to populations with high exposure levels.Altogether, these findings indicate that BPA exposure plays a role in influencing the outcomes of chemotherapy.

Futur e dir ections
Future directions of research could focus on identifying more accurate measurements of human exposure to BPA.Typically, human exposure is reported as levels of conjugated BPA in either urine or serum.However, studies have shown that biomonitoring of BPA via blood or urine may underestimate the total body burden.For example, BPA can also be excreted and detected in sweat and adipose tissue, even in individuals whose urine or serum samples exhibit no traces of BPA ( 11 ,81 ).Moreover, BPA has also been shown to have a disproportionate affinity for fat as compared to other tissues including kidney and muscle tissue ( 82 ).These findings indicate a need for further investigation of BPA accumulation in the human body as well as its potential to exert longitudinal effects on genomic stability .Additionally , given that BPA, like other estrogenic chemicals, exhibits a non-monotonic dose relationship, establishing whether higher levels of exposure for a shorter duration mimic the lifetime exposure in most humans is imperative to consider the relevance of findings obtained in cell-culture studies and how they may translate to human health.
Moreover, there is a need to further investigate the effect of BPA-mediated ER activation on promoting genomic instability.While it has been shown that BPA exerts a greater DNA damaging effect on cells expressing the estrogen receptor, little is known about the mechanism in which this occurs.Investigating BPA-mediated gene regulation via ER activation, particularly genes which play a role in exacerbating BPA-mediated DNA damage, such as MRE11 and EXO1, is necessary to further define this mechanism.Additionally, because ER activation also promotes cellular proliferation, it is important to uncover whether BPA mediated-ER activation plays a role in further hindering mitotic progression or results in suppression of checkpoint pathways, leading to further chromosomal instability.It would also be interesting to compare the role of BPA in cancer initiation in ER+ and ERmodels, as BPA also seems to enhance cellular proliferation in a manner independent of ER activation.
Another aspect of note is further defining the mechanism by which BPA promotes ssDNA gap and dsDNA break formation, independent of ER activation.While it has been shown that BPA mutational signatures include point mutations as well as chromosomal aberrations, published work has mainly focused on the role of BPA in promoting nascent strand ss-DNA gaps via PrimPol mediated repriming.It would also be interesting to investigate whether BPA lesions on DNA are able to be replicated through by TLS polymerases as well as the mechanism in which BPA-derived ssDNA gaps are filled to suppress their conversion into dsDNA breaks.
In addition, because BPA is able to form DNA adducts, it may exhibit a more pronounced genotoxic effect when cells are proliferating.Moreover, BPA exposure has also been shown to promote the proliferation of cells, potentially exacerbating this phenomenon.The intersection between increased cellular proliferation and accumulation of genotoxicity as it relates to BPA exposure has yet to be fully understood.Investigating the relationship between these variables in both ER-dependent and independent backgrounds is necessary to fully uncover the role of BPA in promoting genotoxicity and ultimately carcinogenesis.
Finally, future directions also include identifying genes that, when lost, predispose individuals to BPA-mediated DNA damage and cellular sensitivity.Methods to investigate this include CRISPR and shRNA screens.Identifying populations that are more vulnerable to BPA-mediated genomic instability could help establish safety guidelines that prevent these individuals from excess exposure to BPA, particularly those employed in the plastic manufacturing industry.Overall, further investigation of the effects of BPA on genomic instability is crucial to understand how this chemical plays a role in promoting carcinogenesis.

Conclusion
While BPA exposure has been associated with a wide variety of adverse health effects such as abnormalities in reproductive organ function, placental dysfunction, precocious puberty, and neurological impairment, its role as a carcinogen is less defined.Via either formation of adducts with DNA or activation of estrogen receptor signaling, recent studies suggested that BPA promotes genomic instability at both point mutations and chromosome structure levels, as well as oxidative damage to lipids and proteins.Through transcriptional regulation, BPA also promotes cellular proliferation and migration, resulting in decreased tumor latency and increased tumor burden.Moreover, BPA's ability to impact chromatin structure and promote the evasion of apoptosis also leads to an increase in resistance to chemotherapy, a key clinical problem.While more work must be done to uncover the potential role of BPA in cancer formation and define its function as a carcinogen, recent studies summarized here have shown that BPA exposure results in genomic instability, which is considered an enabling characteristic of carcinogenesis.Knowledge about the physiological effects of BPA on DNA damage and cancer formation can be used to better define the mechanisms that guide DNA damage tolerance as well as guide treatment decisions in patients affected by BPA exposure.Finally, identifying the mechanisms by which BPA causes DNA damage will help to define the health risks of BPA and serve to outline measures that may be taken to further decrease human exposure.

Figure 1 .
Figure 1.Str uct ure and metabolization of BP A. After BP A enters the body , it is metaboliz ed and inactiv ated in the liv er b y the addition of glucuronide, forming Bisphenol A glucuronide.This compound is then filtered out of the blood by the kidneys and is excreted in the urine.BPA can also be deconjugated and reactivated by β-glucuronidase.Unconjugated BPA is oxidized by cytochrome P450 (CYP), forming Bisphenol A 3,4-quinone, which has the potential to form adducts with DNA.Created in BioRender.Moldovan, G. (2024) BioRender.com/ o52i455.

Figure 3 .
Figure 3. BPA may promote carcinogenesis through alteration of transcriptional activity.BPA has been shown to upregulate c-myc via ESR1 and HD A C activation.BPA also can activate the GPER / EGFR / ERK and mTOR signal transduction pathways, promoting cellular proliferation and carcinogenesis ( A ). BPA exposure also promotes reduced expression of proapoptotic regulators of the cell cycle including, p53, p21 WAF1 and BAX, enhancing chemoresistance ( B ). BPA has been shown to induce the expression of HOXB9, a homeobox protein known to modify chromatin methylation and promote gene activation ( C ). BPA can also upregulate TBX3 and NANOG, enhancing the self-renewal of stem progenitor cells ( D ).Finally, BPA inhibits expression of E-Cadherin, promoting epithelial to mesenchymal transition, associated with met ast asis ( E ).Created in BioRender.Moldovan, G. (2024) BioRender.com/ s44z602.